pr8  (Sino Biological)


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    Sino Biological pr8
    Heterosubtypic protection against H1 virus by anti–MHCII-MIX vaccination is independent of T cells. ( A and B ) BALB/c mice ( n = 7 per group) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines. Sera from mice of each group were harvested 2 wk after the third vaccination and transferred i.v. to naive mice ( n = 10 per group) before a lethal challenge with 5 × LD 50 <t>PR8.</t> Mice were monitored for weight loss (A) and survival (B). The results are representative of two independent experiments. ( C and D ) BALB/c mice ( n = 10) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines and subsequently were injected every other day from day 12 after the third vaccination with depleting mAbs against CD4 and CD8 T cells or isotype-matched control mAbs. A 5 × LD 50 lethal dose of PR8 was administered at day 14, and the mice were monitored for weight loss (C) and survival (D). Efficient depletion was confirmed by staining of splenocytes. * p
    Pr8, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pr8/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pr8 - by Bioz Stars, 2021-07
    94/100 stars

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    1) Product Images from "Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza"

    Article Title: Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1701088

    Heterosubtypic protection against H1 virus by anti–MHCII-MIX vaccination is independent of T cells. ( A and B ) BALB/c mice ( n = 7 per group) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines. Sera from mice of each group were harvested 2 wk after the third vaccination and transferred i.v. to naive mice ( n = 10 per group) before a lethal challenge with 5 × LD 50 PR8. Mice were monitored for weight loss (A) and survival (B). The results are representative of two independent experiments. ( C and D ) BALB/c mice ( n = 10) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines and subsequently were injected every other day from day 12 after the third vaccination with depleting mAbs against CD4 and CD8 T cells or isotype-matched control mAbs. A 5 × LD 50 lethal dose of PR8 was administered at day 14, and the mice were monitored for weight loss (C) and survival (D). Efficient depletion was confirmed by staining of splenocytes. * p
    Figure Legend Snippet: Heterosubtypic protection against H1 virus by anti–MHCII-MIX vaccination is independent of T cells. ( A and B ) BALB/c mice ( n = 7 per group) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines. Sera from mice of each group were harvested 2 wk after the third vaccination and transferred i.v. to naive mice ( n = 10 per group) before a lethal challenge with 5 × LD 50 PR8. Mice were monitored for weight loss (A) and survival (B). The results are representative of two independent experiments. ( C and D ) BALB/c mice ( n = 10) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines and subsequently were injected every other day from day 12 after the third vaccination with depleting mAbs against CD4 and CD8 T cells or isotype-matched control mAbs. A 5 × LD 50 lethal dose of PR8 was administered at day 14, and the mice were monitored for weight loss (C) and survival (D). Efficient depletion was confirmed by staining of splenocytes. * p

    Techniques Used: Mouse Assay, Injection, Staining

    Vaccination with anti–MHCII-MIX confers heterosubtypic protection against H1 influenza viruses not included in the vaccine mixture. ( A – F ) BALB/c mice ( n = 6 per group) were immunized three times (weeks 0, 4, and 12, arrows) with 25 μg of DNA/EP of the indicated vaccines. As positive controls, anti–MHCII-H1(PR8) and anti–MHCII-H1(Cal07) were included. Serum IgG Abs were measured against PR8 (A) or Cal07 (B) in ELISAs (mean ± SEM). Microneutralization assays were performed on sera harvested 2 wk after the third vaccination with PR8 (C) and Cal07 (D). The dashed line indicates 50% threshold for positive neutralization. Two weeks after the third vaccination (above), mice were challenged with a lethal dose of Cal07 and monitored for weight loss (± SEM) (E) and survival (F). ( G and H ) In a new experiment, mice were vaccinated at weeks 0, 4, and 12 (as above) and challenged with a lethal dose of influenza PR8 2 wk after the third vaccination ( n = 10 per group [black], n = 6 per group [gray]). Mice were monitored for weight loss (± SEM) (G) and survival (H). * p
    Figure Legend Snippet: Vaccination with anti–MHCII-MIX confers heterosubtypic protection against H1 influenza viruses not included in the vaccine mixture. ( A – F ) BALB/c mice ( n = 6 per group) were immunized three times (weeks 0, 4, and 12, arrows) with 25 μg of DNA/EP of the indicated vaccines. As positive controls, anti–MHCII-H1(PR8) and anti–MHCII-H1(Cal07) were included. Serum IgG Abs were measured against PR8 (A) or Cal07 (B) in ELISAs (mean ± SEM). Microneutralization assays were performed on sera harvested 2 wk after the third vaccination with PR8 (C) and Cal07 (D). The dashed line indicates 50% threshold for positive neutralization. Two weeks after the third vaccination (above), mice were challenged with a lethal dose of Cal07 and monitored for weight loss (± SEM) (E) and survival (F). ( G and H ) In a new experiment, mice were vaccinated at weeks 0, 4, and 12 (as above) and challenged with a lethal dose of influenza PR8 2 wk after the third vaccination ( n = 10 per group [black], n = 6 per group [gray]). Mice were monitored for weight loss (± SEM) (G) and survival (H). * p

    Techniques Used: Mouse Assay, Neutralization

    Characterization of vaccine proteins. ( A ) The MHCII-targeted vaccine proteins consist of a targeting unit (scFv) that is specific for MHC class II (I), a dimerization unit containing a shortened hinge and C H 3 from human γ3 (II), and an antigenic unit encoding one of the six indicated HAs (III). Nontargeted controls have been prepared by replacement of the targeting unit with an scFv that is specific for the hapten NIP. ( B ) Supernatants from HEK 293E cells transfected with the DNA plasmids encoding the indicated vaccines were analyzed in sandwich ELISAs. An MHCII-targeted vaccine encoding HA from PR8 (anti–MHCII-H1) was included as positive control.
    Figure Legend Snippet: Characterization of vaccine proteins. ( A ) The MHCII-targeted vaccine proteins consist of a targeting unit (scFv) that is specific for MHC class II (I), a dimerization unit containing a shortened hinge and C H 3 from human γ3 (II), and an antigenic unit encoding one of the six indicated HAs (III). Nontargeted controls have been prepared by replacement of the targeting unit with an scFv that is specific for the hapten NIP. ( B ) Supernatants from HEK 293E cells transfected with the DNA plasmids encoding the indicated vaccines were analyzed in sandwich ELISAs. An MHCII-targeted vaccine encoding HA from PR8 (anti–MHCII-H1) was included as positive control.

    Techniques Used: Transfection, Positive Control

    2) Product Images from "Combined use of live-attenuated and inactivated influenza vaccines to enhance heterosubtypic protection"

    Article Title: Combined use of live-attenuated and inactivated influenza vaccines to enhance heterosubtypic protection

    Journal: Virology

    doi: 10.1016/j.virol.2018.09.007

    Virus load in infected lung determined by TCID 50 assays. Vaccinated mice infected with H1N1/PR8 (A) or H3N2/HK68 (B) were sacrificed at 3 and 7 dpi (n=3 per group). Detection limit (DL) is as shown. Data represent Mean ± SD; *P
    Figure Legend Snippet: Virus load in infected lung determined by TCID 50 assays. Vaccinated mice infected with H1N1/PR8 (A) or H3N2/HK68 (B) were sacrificed at 3 and 7 dpi (n=3 per group). Detection limit (DL) is as shown. Data represent Mean ± SD; *P

    Techniques Used: Infection, Mouse Assay

    Heterologous protection induced by different vaccination regimens. 3 weeks after vaccination, mice were challenged (i.n.) with a 10MLD 50 dose of H1N1/PR8 (A) or H3N2/HK68 (B), or a MLD 90 dose of HAPI H7N7/NL03 (C) as indicated. Survival rate were assessed daily for a period of 14 days (n≥10 per group). Log-rank test: *P
    Figure Legend Snippet: Heterologous protection induced by different vaccination regimens. 3 weeks after vaccination, mice were challenged (i.n.) with a 10MLD 50 dose of H1N1/PR8 (A) or H3N2/HK68 (B), or a MLD 90 dose of HAPI H7N7/NL03 (C) as indicated. Survival rate were assessed daily for a period of 14 days (n≥10 per group). Log-rank test: *P

    Techniques Used: Mouse Assay

    Recalled immune responses against heterologous/heterosubtypic IAV infection in sequential-vaccinated mice. At 7 dpi, CD8 + T cell responses were determined from splenocytes by ICS assay. Representative FACS plots of type-1 cytokines against NP 147–155 produced in CD8 + T cells (A, left) and percentages of Tc1 antigen-specific cytokines-producing CD8 + T cells (A, right) post H1N1/PR8 infection. Representative FACS plots of type-1 cytokines against NP 147–155 produced in CD8 + T cells (B, left) and percentages of Tc1 antigen-specific cytokines-producing CD8 + T cells (B, right) post H3N2/HK68 infection. Q1, IFN-γ+; Q3, TNF-α+; Q2, IFN-γ+TNF-α+. Data represent Mean ± SD; *P
    Figure Legend Snippet: Recalled immune responses against heterologous/heterosubtypic IAV infection in sequential-vaccinated mice. At 7 dpi, CD8 + T cell responses were determined from splenocytes by ICS assay. Representative FACS plots of type-1 cytokines against NP 147–155 produced in CD8 + T cells (A, left) and percentages of Tc1 antigen-specific cytokines-producing CD8 + T cells (A, right) post H1N1/PR8 infection. Representative FACS plots of type-1 cytokines against NP 147–155 produced in CD8 + T cells (B, left) and percentages of Tc1 antigen-specific cytokines-producing CD8 + T cells (B, right) post H3N2/HK68 infection. Q1, IFN-γ+; Q3, TNF-α+; Q2, IFN-γ+TNF-α+. Data represent Mean ± SD; *P

    Techniques Used: Infection, Mouse Assay, FACS, Produced

    3) Product Images from "αv Integrins regulate germinal center B cell responses through noncanonical autophagy"

    Article Title: αv Integrins regulate germinal center B cell responses through noncanonical autophagy

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI99597

    Deletion of α v enhances antibody response to influenza virus. ( A and B ) Serum anti-PR/8 IgG titers in control and α v -CD19 mice immunized with 10 μg of inactivated H1N1 PR/8 ( A ) and ( B ) boosted at day 57 with 5 μg inactivated PR/8. ( C ) PR/8-specific plasma cells enumerated by ELISpot in BM cells from control (Con) and α v -CD19 mice harvested at day 7 after boost. ( D ) HAI activity in sera from control and α v -CD19 mice at day 21 after PR/8 immunization. ( E and F ) Serum antibody titers against HA from H1N1 PR/8 ( E ) or H1N1 Cal/09 ( F ) in control and α v -CD19 mice at day 7 after boost with inactivated PR/8. ( G ) Anti-Cal/09 HA titer normalized to anti-PR/8 HA titer. ( H and I ) Serum antibody titers against HA from PR/8 ( H ) or Cal/09 ( I ) in control and α v -CD19 at day 51 after immunization with inactivated PR/8 (10 μg) in imiquimod-SE (10 μg). ( J ) Anti-Cal/09 HA titer normalized to anti-PR/8 HA titer for mice immunized with PR/8 in imiquimod-SE. ( K ) Survival of control and α v -CD19 mice following intranasal infection with PR/8 ( n ≥ 5 mice/group). ( L ) Anti-PR8 HA titers from surviving mice at day 7 after infection. All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test for antibody titers or Mantel-Cox test for survival curves).* P
    Figure Legend Snippet: Deletion of α v enhances antibody response to influenza virus. ( A and B ) Serum anti-PR/8 IgG titers in control and α v -CD19 mice immunized with 10 μg of inactivated H1N1 PR/8 ( A ) and ( B ) boosted at day 57 with 5 μg inactivated PR/8. ( C ) PR/8-specific plasma cells enumerated by ELISpot in BM cells from control (Con) and α v -CD19 mice harvested at day 7 after boost. ( D ) HAI activity in sera from control and α v -CD19 mice at day 21 after PR/8 immunization. ( E and F ) Serum antibody titers against HA from H1N1 PR/8 ( E ) or H1N1 Cal/09 ( F ) in control and α v -CD19 mice at day 7 after boost with inactivated PR/8. ( G ) Anti-Cal/09 HA titer normalized to anti-PR/8 HA titer. ( H and I ) Serum antibody titers against HA from PR/8 ( H ) or Cal/09 ( I ) in control and α v -CD19 at day 51 after immunization with inactivated PR/8 (10 μg) in imiquimod-SE (10 μg). ( J ) Anti-Cal/09 HA titer normalized to anti-PR/8 HA titer for mice immunized with PR/8 in imiquimod-SE. ( K ) Survival of control and α v -CD19 mice following intranasal infection with PR/8 ( n ≥ 5 mice/group). ( L ) Anti-PR8 HA titers from surviving mice at day 7 after infection. All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test for antibody titers or Mantel-Cox test for survival curves).* P

    Techniques Used: Mouse Assay, Enzyme-linked Immunospot, Activity Assay, Infection, MANN-WHITNEY

    4) Product Images from "αv Integrins regulate germinal center B cell responses through noncanonical autophagy"

    Article Title: αv Integrins regulate germinal center B cell responses through noncanonical autophagy

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI99597

    Deletion of α v enhances antibody response to influenza virus. ( A and B ) Serum anti-PR/8 IgG titers in control and α v -CD19 mice immunized with 10 μg of inactivated H1N1 PR/8 ( A ) and ( B ) boosted at day 57 with 5 μg inactivated PR/8. ( C ) PR/8-specific plasma cells enumerated by ELISpot in BM cells from control (Con) and α v -CD19 mice harvested at day 7 after boost. ( D ) HAI activity in sera from control and α v -CD19 mice at day 21 after PR/8 immunization. ( E and F ) Serum antibody titers against HA from H1N1 PR/8 ( E ) or H1N1 Cal/09 ( F ) in control and α v -CD19 mice at day 7 after boost with inactivated PR/8. ( G ) Anti-Cal/09 HA titer normalized to anti-PR/8 HA titer. ( H and I ) Serum antibody titers against HA from PR/8 ( H ) or Cal/09 ( I ) in control and α v -CD19 at day 51 after immunization with inactivated PR/8 (10 μg) in imiquimod-SE (10 μg). ( J ) Anti-Cal/09 HA titer normalized to anti-PR/8 HA titer for mice immunized with PR/8 in imiquimod-SE. ( K ) Survival of control and α v -CD19 mice following intranasal infection with PR/8 ( n ≥ 5 mice/group). ( L ) Anti-PR8 HA titers from surviving mice at day 7 after infection. All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test for antibody titers or Mantel-Cox test for survival curves).* P
    Figure Legend Snippet: Deletion of α v enhances antibody response to influenza virus. ( A and B ) Serum anti-PR/8 IgG titers in control and α v -CD19 mice immunized with 10 μg of inactivated H1N1 PR/8 ( A ) and ( B ) boosted at day 57 with 5 μg inactivated PR/8. ( C ) PR/8-specific plasma cells enumerated by ELISpot in BM cells from control (Con) and α v -CD19 mice harvested at day 7 after boost. ( D ) HAI activity in sera from control and α v -CD19 mice at day 21 after PR/8 immunization. ( E and F ) Serum antibody titers against HA from H1N1 PR/8 ( E ) or H1N1 Cal/09 ( F ) in control and α v -CD19 mice at day 7 after boost with inactivated PR/8. ( G ) Anti-Cal/09 HA titer normalized to anti-PR/8 HA titer. ( H and I ) Serum antibody titers against HA from PR/8 ( H ) or Cal/09 ( I ) in control and α v -CD19 at day 51 after immunization with inactivated PR/8 (10 μg) in imiquimod-SE (10 μg). ( J ) Anti-Cal/09 HA titer normalized to anti-PR/8 HA titer for mice immunized with PR/8 in imiquimod-SE. ( K ) Survival of control and α v -CD19 mice following intranasal infection with PR/8 ( n ≥ 5 mice/group). ( L ) Anti-PR8 HA titers from surviving mice at day 7 after infection. All data points represent individual mice with mean shown. P values of less than 0.05 are shown (Mann-Whitney-Wilcoxon test for antibody titers or Mantel-Cox test for survival curves).* P

    Techniques Used: Mouse Assay, Enzyme-linked Immunospot, Activity Assay, Infection, MANN-WHITNEY

    Related Articles

    Recombinant:

    Article Title: Induction of Cross-Reactive and Protective Antibody Responses After DNA Vaccination With MHCII-Targeted Stem Domain From Influenza Hemagglutinin
    Article Snippet: ELISA for Detection of Serum Antibodies Blood was harvested by puncture of the saphenous vein, and sera were collected by centrifugation. .. Ninety-six-well plates (Costar 3590) were coated with inactivated A/PR/8/34 (H1N1) (PR8) virus (1:1600) (virus supplied from Charles River, USA, with HA titer 1:65,536 in 0.05 ml) in PBS azide, or with recombinant HA protein (1 μg/ml) from one of the following viruses: PR8 (11684-V08H, Sino Biological Inc., USA), A/Hong Kong/1/1968 (H3N2) (40116-V08H1, Sino Biological Inc.), A/Hong Kong/483/97 (H5N1) (11689-V08H, Sino Biological Inc.), A/Shanghai/1/2013 (H7N9) (40104-V08H, Sino Biological Inc.), or A/Hong Kong/1073/99 (H9N2) (11229-V08H, Sino Biological Inc.). .. Next, plates were incubated with either alkaline phosphatase conjugated anti-mouse IgG (A1418, Sigma-Aldrich, Germany), biotinylated anti-IgG1a (553599, BD Pharmingen), or biotinylated anti-IgG2aa (553502, BD Pharmingen), and developed as above.

    Article Title: Targeting of HA to chemokine receptors induces strong and cross-reactive T cell responses after DNA vaccination in pigs.
    Article Snippet: Serum ELISASera were isolated from blood by centrifugation. .. ELISA 96-well plates (Costar 3590, Sigma-Aldrich) were coated with either recombinant HA from PR8 (11684-V08H, Sino Biological Inc., North Wales, PA, US), rec. .. HA from A/Anhui/1/2005 (H5N1) (11048- V08H1, Sino Biological Inc.), rec.

    Article Title: Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza
    Article Snippet: .. ELISA 96-well plates (Costar 3590) were coated with one of the following recombinant influenza HA proteins: PR8 (11684-V08H; Sino Biological), A/Hong Kong/483/97 (H5N1) (11689-V08H; Sino Biological), A/northern shoveler/California/HKWF115/07 (H6N1) (MBS434125; MyBioSource, San Diego, CA, or 11723-V08H; Sino Biological), A/pintail duck/Alberta/114/1979 (H8N4) (11722-V08H; Sino Biological), A/Hong Kong/1073/99 (H9N2) (11229-V08H; Sino Biological), A/duck/Yangzhou/906/2002 (H11N2) (11705-V08H; Sino Biological), A/black-headed gull/Netherlands/1/00 (H13N8) (11721-V08H; Sino Biological), A/Shanghai/1/2013 (H7N9) (40104-V08B; Sino Biological), or A/Hong Kong/1/1968 (H3N2) (40116-V08B; Sino Biological). .. Alternatively, plates were coated with inactivated virus: PR8 (1:1600; Charles River, Wilmington, MA) or Cal07 (Pandemrix; 1:50; GlaxoSmithKline, Brentford, U.K.).

    Article Title: Induction of Cross-Reactive and Protective Antibody Responses After DNA Vaccination With MHCII-Targeted Stem Domain From Influenza Hemagglutinin
    Article Snippet: .. The ELISAs were set up with coats of HA protein from influenza PR8 (11684-V08H, Sino Biological Inc., USA) or A/Hong Kong/483/97 (H5N1) (11689-V08H, Sino Biological Inc.), or ovalbumin (OVA) (A5503, Sigma), or by coating with Phox-BSA and recombinant proteins expressing Phox-specific scFv linked to the HA stem. .. Detection was performed with either alkaline phosphatase-conjugated anti-mouse IgG (A1418 Sigma-Aldrich) or biotinylated anti-IgM (553515, BD Pharmingen) and streptavidin alkaline phosphatase (1:3000, GE Healthcare).

    Article Title: Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses
    Article Snippet: The blocking protein for ELISA assays was BSA (Sigma). .. The recombinant PR8 HA protein was purchased from Sino Biological Inc. (catalog number 11684-V08H) and was reconstituted in sterile PBS. .. The MF59 adjuvant (AddaVax) was purchased from InvivoGen.

    Article Title: Toxicity and Immunogenicity of a Tardigrade Cytosolic Abundant Heat Soluble Protein in Mice
    Article Snippet: The first group was given Lactated Ringer’s solution as a negative control. .. The next two groups were given low (1.25 g/L) or high (15 g/L) doses of CAHS D. The final group was given 0.05 g/L recombinant hemagglutinin protein (HA) from the influenza A H1N1 (A/Puerto Rico/8/1934) flu virus (Sino Biological 11684-V08H). ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: Targeting of HA to chemokine receptors induces strong and cross-reactive T cell responses after DNA vaccination in pigs.
    Article Snippet: Serum ELISASera were isolated from blood by centrifugation. .. ELISA 96-well plates (Costar 3590, Sigma-Aldrich) were coated with either recombinant HA from PR8 (11684-V08H, Sino Biological Inc., North Wales, PA, US), rec. .. HA from A/Anhui/1/2005 (H5N1) (11048- V08H1, Sino Biological Inc.), rec.

    Article Title: Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza
    Article Snippet: .. ELISA 96-well plates (Costar 3590) were coated with one of the following recombinant influenza HA proteins: PR8 (11684-V08H; Sino Biological), A/Hong Kong/483/97 (H5N1) (11689-V08H; Sino Biological), A/northern shoveler/California/HKWF115/07 (H6N1) (MBS434125; MyBioSource, San Diego, CA, or 11723-V08H; Sino Biological), A/pintail duck/Alberta/114/1979 (H8N4) (11722-V08H; Sino Biological), A/Hong Kong/1073/99 (H9N2) (11229-V08H; Sino Biological), A/duck/Yangzhou/906/2002 (H11N2) (11705-V08H; Sino Biological), A/black-headed gull/Netherlands/1/00 (H13N8) (11721-V08H; Sino Biological), A/Shanghai/1/2013 (H7N9) (40104-V08B; Sino Biological), or A/Hong Kong/1/1968 (H3N2) (40116-V08B; Sino Biological). .. Alternatively, plates were coated with inactivated virus: PR8 (1:1600; Charles River, Wilmington, MA) or Cal07 (Pandemrix; 1:50; GlaxoSmithKline, Brentford, U.K.).

    Expressing:

    Article Title: Induction of Cross-Reactive and Protective Antibody Responses After DNA Vaccination With MHCII-Targeted Stem Domain From Influenza Hemagglutinin
    Article Snippet: .. The ELISAs were set up with coats of HA protein from influenza PR8 (11684-V08H, Sino Biological Inc., USA) or A/Hong Kong/483/97 (H5N1) (11689-V08H, Sino Biological Inc.), or ovalbumin (OVA) (A5503, Sigma), or by coating with Phox-BSA and recombinant proteins expressing Phox-specific scFv linked to the HA stem. .. Detection was performed with either alkaline phosphatase-conjugated anti-mouse IgG (A1418 Sigma-Aldrich) or biotinylated anti-IgM (553515, BD Pharmingen) and streptavidin alkaline phosphatase (1:3000, GE Healthcare).

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    Sino Biological pr8 np protein
    “Late” CD4 + T cell help shapes respiratory mucosal memory CD8 + and B cell immunity. WT mice were infected with <t>PR8</t> and treated with ctl IgG or α-CD4. a , The efficiency of CD4 + T cell depletion in the lung or spleen. b , Representative image of lung section stained with H E. Black arrows indicate tertial lymphoid structure. c , Representative dot plot and cell numbers of lung germinal center B (B GC ) cells. d , Frequencies of lung circulating (CD45 i.v. + ) or parenchymal (CD45 i.v. - ) CD8 + T or B cells in mice treated with control IgG or α-CD4. e , Influenza HA-specific B cells (HA-B) in the lungs were identified through HA antigen staining. f , Lung tissue or circulating CD8 + NP 366-374 or PA 224-233 T cells were identified through CD45 i.v. staining and analyzed by flow cytometry. g , Lung circulating CD8 + NP 366-374 or CD8 + PA 224-233 T cells were enumerated. i , Histogram of CD103 expression or frequency of CD103 + cells within CD8 + CD69 + NP 366-374 T RM cells. h, j , Gating strategies of CD8 + T RM ( h ) or B RM ( j ) cells in the lung. Representative data were from at least two independent experiments (4-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.
    Pr8 Np Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pr8 np protein/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pr8 np protein - by Bioz Stars, 2021-07
    95/100 stars
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    94
    Sino Biological pr8
    Heterosubtypic protection against H1 virus by anti–MHCII-MIX vaccination is independent of T cells. ( A and B ) BALB/c mice ( n = 7 per group) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines. Sera from mice of each group were harvested 2 wk after the third vaccination and transferred i.v. to naive mice ( n = 10 per group) before a lethal challenge with 5 × LD 50 <t>PR8.</t> Mice were monitored for weight loss (A) and survival (B). The results are representative of two independent experiments. ( C and D ) BALB/c mice ( n = 10) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines and subsequently were injected every other day from day 12 after the third vaccination with depleting mAbs against CD4 and CD8 T cells or isotype-matched control mAbs. A 5 × LD 50 lethal dose of PR8 was administered at day 14, and the mice were monitored for weight loss (C) and survival (D). Efficient depletion was confirmed by staining of splenocytes. * p
    Pr8, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pr8/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pr8 - by Bioz Stars, 2021-07
    94/100 stars
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    Full length Clone DNA of Influenza A H1N1 A Puerto Rico 8 34 Hemagglutinin with C terminal GFPSpark tag
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    Image Search Results


    “Late” CD4 + T cell help shapes respiratory mucosal memory CD8 + and B cell immunity. WT mice were infected with PR8 and treated with ctl IgG or α-CD4. a , The efficiency of CD4 + T cell depletion in the lung or spleen. b , Representative image of lung section stained with H E. Black arrows indicate tertial lymphoid structure. c , Representative dot plot and cell numbers of lung germinal center B (B GC ) cells. d , Frequencies of lung circulating (CD45 i.v. + ) or parenchymal (CD45 i.v. - ) CD8 + T or B cells in mice treated with control IgG or α-CD4. e , Influenza HA-specific B cells (HA-B) in the lungs were identified through HA antigen staining. f , Lung tissue or circulating CD8 + NP 366-374 or PA 224-233 T cells were identified through CD45 i.v. staining and analyzed by flow cytometry. g , Lung circulating CD8 + NP 366-374 or CD8 + PA 224-233 T cells were enumerated. i , Histogram of CD103 expression or frequency of CD103 + cells within CD8 + CD69 + NP 366-374 T RM cells. h, j , Gating strategies of CD8 + T RM ( h ) or B RM ( j ) cells in the lung. Representative data were from at least two independent experiments (4-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: “Late” CD4 + T cell help shapes respiratory mucosal memory CD8 + and B cell immunity. WT mice were infected with PR8 and treated with ctl IgG or α-CD4. a , The efficiency of CD4 + T cell depletion in the lung or spleen. b , Representative image of lung section stained with H E. Black arrows indicate tertial lymphoid structure. c , Representative dot plot and cell numbers of lung germinal center B (B GC ) cells. d , Frequencies of lung circulating (CD45 i.v. + ) or parenchymal (CD45 i.v. - ) CD8 + T or B cells in mice treated with control IgG or α-CD4. e , Influenza HA-specific B cells (HA-B) in the lungs were identified through HA antigen staining. f , Lung tissue or circulating CD8 + NP 366-374 or PA 224-233 T cells were identified through CD45 i.v. staining and analyzed by flow cytometry. g , Lung circulating CD8 + NP 366-374 or CD8 + PA 224-233 T cells were enumerated. i , Histogram of CD103 expression or frequency of CD103 + cells within CD8 + CD69 + NP 366-374 T RM cells. h, j , Gating strategies of CD8 + T RM ( h ) or B RM ( j ) cells in the lung. Representative data were from at least two independent experiments (4-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Staining, Flow Cytometry, Expressing, Two Tailed Test

    T RH cells help CD8 + T and B cells through IL-21 or CD40L dependent mechanisms. a , Expression of Il21 gene in sorted IL-21-VFP Hi, IL-21-VFP Low or IL-21-VFP - CD4 + T cells from the lung tissue at 21 d.p.i. (pooled from 8 mice) b , Expression of Il21 in the whole lung from Bcl6 fl/fl or Bcl6 ΔT mice at 15 or 42 d.p.i. c , Frequencies or cell numbers of lung B GC or HA-specific B cells from PR8 infected WT mice treated with ctl-IgG or α-IL-21R (Intranasal route). d , Heat map of IL-21 signaling-related genes from sorted lung CD8 + NP 366-374 or PA 224-233 T RM or splenic CD8 + NP 366-374 or PA 224-233 T MEM determined by Nanostring at 38 d.p.i. as reported previously 40 . e , Geometric M.F.I. of BATF expression in lung CD8 + NP 366-374, CD8 + PA 224-233 T RM , splenic CD8 + NP 366-374 or PA 224-233 T MEM cells from PR8 infected mice at 35 d.p.i. f , CD40L expression in the NP 311-325 T RH or non-T RH cells following PMA/Ionomycin stimulation at 28 d.p.i. g , Schematics of the summary. In b-c and e-f , representative data were from at least two independent experiments (3-5 mice per group). P values were calculated by unpaired (b and c) or paired (f) two-tailed Student’s t-test. P value in e was analyzed by one-way ANOVA.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: T RH cells help CD8 + T and B cells through IL-21 or CD40L dependent mechanisms. a , Expression of Il21 gene in sorted IL-21-VFP Hi, IL-21-VFP Low or IL-21-VFP - CD4 + T cells from the lung tissue at 21 d.p.i. (pooled from 8 mice) b , Expression of Il21 in the whole lung from Bcl6 fl/fl or Bcl6 ΔT mice at 15 or 42 d.p.i. c , Frequencies or cell numbers of lung B GC or HA-specific B cells from PR8 infected WT mice treated with ctl-IgG or α-IL-21R (Intranasal route). d , Heat map of IL-21 signaling-related genes from sorted lung CD8 + NP 366-374 or PA 224-233 T RM or splenic CD8 + NP 366-374 or PA 224-233 T MEM determined by Nanostring at 38 d.p.i. as reported previously 40 . e , Geometric M.F.I. of BATF expression in lung CD8 + NP 366-374, CD8 + PA 224-233 T RM , splenic CD8 + NP 366-374 or PA 224-233 T MEM cells from PR8 infected mice at 35 d.p.i. f , CD40L expression in the NP 311-325 T RH or non-T RH cells following PMA/Ionomycin stimulation at 28 d.p.i. g , Schematics of the summary. In b-c and e-f , representative data were from at least two independent experiments (3-5 mice per group). P values were calculated by unpaired (b and c) or paired (f) two-tailed Student’s t-test. P value in e was analyzed by one-way ANOVA.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Expressing, Mouse Assay, Infection, Two Tailed Test

    Optimal T RH formation requires lung B cells and iBALT formation WT ( a-d ) mice were infected with PR8 and treated with ctl IgG, α-CD20 ( b ) or LTβR-Ig ( c and d ) (weekly starting at 14 d.p.i.) in the presence of daily injection of FTY-720 (13-27 d.p.i.). a , Experimental scheme. b and d , Representative dot plot and cell numbers of influenza-specific NP 311-325 lung T RH, lung non-T RH or splenic T FH cells. c , Representative image from lung tissue section stained with B220/GL-7 following ctl IgG or LTβR-Ig treatment. In c , the representative image was from at least two independent experiments (3-4 mice per group). In b , representative data were from at least two independent experiments (4-5 mice per group). In d , data were pooled from two independent experiments (3-4 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Optimal T RH formation requires lung B cells and iBALT formation WT ( a-d ) mice were infected with PR8 and treated with ctl IgG, α-CD20 ( b ) or LTβR-Ig ( c and d ) (weekly starting at 14 d.p.i.) in the presence of daily injection of FTY-720 (13-27 d.p.i.). a , Experimental scheme. b and d , Representative dot plot and cell numbers of influenza-specific NP 311-325 lung T RH, lung non-T RH or splenic T FH cells. c , Representative image from lung tissue section stained with B220/GL-7 following ctl IgG or LTβR-Ig treatment. In c , the representative image was from at least two independent experiments (3-4 mice per group). In b , representative data were from at least two independent experiments (4-5 mice per group). In d , data were pooled from two independent experiments (3-4 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Injection, Staining, Two Tailed Test

    IL-21 or CD40L-dependent T RH help to CD8 + or B cells. a-b , IL-21-VFP reporter mice were infected with PR8. a , IL-21-VFP expressing cells in the lungs or spleens were identified by flow cytometry at 28 d.p.i. b , Representative dot plot of IL-21 Hi or IL-21 Low CD4 + T cells that were PD-1 Hi FR4 Hi . c-f , WT mice were infected PR8 with or without IL-21R blockade through intraperitoneal (I.P.) route starting at 14 d.p.i. in the presence of FTY-720 administration (13-34 d.p.i.). c , Experimental scheme. Cell numbers of lung parenchymal B GC ( d ), HA-specific B RM ( e ) and CD8 + CD69 + NP 366-374 or CD8 + CD69 + PA 224-233 T RM cells ( f ). g-k , WT mice were infected with PR8 with or without IL-21R blockade through intranasal (I.N.) route at 14 d.p.i. g , Experimental scheme. h-i , Frequencies ( h ) or cell numbers ( i ) of lung tissue CD8 + CD69 + NP 366-374, CD8 + CD69 + PA 224-233 T RM , splenic CD8 + NP 366-374 or PA 224- 233 memory T cells (T MEM ) at 42 d.p.i. j , Percentage of apoptotic cells were identified by active caspase 3/7-FLICA staining within lung CD8 + NP 366-374 T RM or splenic CD8 + NP 366-374 T MEM at 28 d.p.i. k , percentages of proliferating cells were identified by Ki67 staining within lung CD8 + NP 366-374 T RM or splenic CD8 + NP 366-374 T MEM at 28 d.p.i. l , Representative histogram of BATF expression in lung CD8 + NP 366-374 or CD8 + PA 224-233 T RM of mice received with ctl IgG or α-IL21R at 35 d.p.i. m , Representative histogram of CD40L expression in influenza-specific lung T RH or non-T RH at 28 d.p.i. n-o , WT mice were infected with PR8 and received ctl IgG or α-CD40L weekly (I.P. route) starting at 14 d.p.i. in the presence of daily FTY-720 administration (13-34 d.p.i.). Representative dot plot or cell numbers B GC ( n ) and HA-specific B RM ( o ) cells at 35 d.p.i. In a-b and j-m , representative data were from at least two independent experiments (4-5 mice per group). In d, f-i and n-o , data were pooled from two independent experiments (3-5 mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: IL-21 or CD40L-dependent T RH help to CD8 + or B cells. a-b , IL-21-VFP reporter mice were infected with PR8. a , IL-21-VFP expressing cells in the lungs or spleens were identified by flow cytometry at 28 d.p.i. b , Representative dot plot of IL-21 Hi or IL-21 Low CD4 + T cells that were PD-1 Hi FR4 Hi . c-f , WT mice were infected PR8 with or without IL-21R blockade through intraperitoneal (I.P.) route starting at 14 d.p.i. in the presence of FTY-720 administration (13-34 d.p.i.). c , Experimental scheme. Cell numbers of lung parenchymal B GC ( d ), HA-specific B RM ( e ) and CD8 + CD69 + NP 366-374 or CD8 + CD69 + PA 224-233 T RM cells ( f ). g-k , WT mice were infected with PR8 with or without IL-21R blockade through intranasal (I.N.) route at 14 d.p.i. g , Experimental scheme. h-i , Frequencies ( h ) or cell numbers ( i ) of lung tissue CD8 + CD69 + NP 366-374, CD8 + CD69 + PA 224-233 T RM , splenic CD8 + NP 366-374 or PA 224- 233 memory T cells (T MEM ) at 42 d.p.i. j , Percentage of apoptotic cells were identified by active caspase 3/7-FLICA staining within lung CD8 + NP 366-374 T RM or splenic CD8 + NP 366-374 T MEM at 28 d.p.i. k , percentages of proliferating cells were identified by Ki67 staining within lung CD8 + NP 366-374 T RM or splenic CD8 + NP 366-374 T MEM at 28 d.p.i. l , Representative histogram of BATF expression in lung CD8 + NP 366-374 or CD8 + PA 224-233 T RM of mice received with ctl IgG or α-IL21R at 35 d.p.i. m , Representative histogram of CD40L expression in influenza-specific lung T RH or non-T RH at 28 d.p.i. n-o , WT mice were infected with PR8 and received ctl IgG or α-CD40L weekly (I.P. route) starting at 14 d.p.i. in the presence of daily FTY-720 administration (13-34 d.p.i.). Representative dot plot or cell numbers B GC ( n ) and HA-specific B RM ( o ) cells at 35 d.p.i. In a-b and j-m , representative data were from at least two independent experiments (4-5 mice per group). In d, f-i and n-o , data were pooled from two independent experiments (3-5 mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Expressing, Flow Cytometry, Hi-C, Staining, Two Tailed Test

    T RH cells help local development of memory CD8 + and B cells. a-c , Bcl6 fl/fl (with ROSA26 LSL-YFP transgene) or Bcl6 ΔCD4ERT2 (with ROSA26 LSL-YFP transgene) mice were infected with X31. Tamoxifen was administrated daily from 12 to 16 d.p.i. a , Experimental scheme of selective deletion of Bcl6 in CD4 + T cells. b, c , YFP expression in blood CD45 + ( b ) or CD4 + T ( c ) cells following tamoxifen injection. d, e , Bcl6 fl/fl or Bcl6 ΔCD4ERT2 mice were infected with PR8. Tamoxifen was administrated daily from 12-16 d.p.i. in the presence of daily FTY720 administration (11-34 d.p.i.). d , Cell numbers of CD45 i.v. - CD4 + T, CD8 + T or B cells at 35 d.p.i. e , Representative dot plot (top) or cell numbers (bottom) of NP 311- 325 T RH or non-T RH cells at 35 d.p.i. f , Cell numbers of lung CD8 + CD69 + NP 366-374 or CD8 + CD69 + PA 224-233 T RM , B GC or HA-specific B RM cells were enumerated from X31-infected Bcl6 fl/fl and Bcl6 ΔCD4ERT2 mice in the present of FTY720 (11-34 d.p.i.) at 35 d.p.i. In a-c and f , representative data were from at least two independent experiments (2-5 mice per group). In d-e , data were pooled from two independent experiments (3 mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: T RH cells help local development of memory CD8 + and B cells. a-c , Bcl6 fl/fl (with ROSA26 LSL-YFP transgene) or Bcl6 ΔCD4ERT2 (with ROSA26 LSL-YFP transgene) mice were infected with X31. Tamoxifen was administrated daily from 12 to 16 d.p.i. a , Experimental scheme of selective deletion of Bcl6 in CD4 + T cells. b, c , YFP expression in blood CD45 + ( b ) or CD4 + T ( c ) cells following tamoxifen injection. d, e , Bcl6 fl/fl or Bcl6 ΔCD4ERT2 mice were infected with PR8. Tamoxifen was administrated daily from 12-16 d.p.i. in the presence of daily FTY720 administration (11-34 d.p.i.). d , Cell numbers of CD45 i.v. - CD4 + T, CD8 + T or B cells at 35 d.p.i. e , Representative dot plot (top) or cell numbers (bottom) of NP 311- 325 T RH or non-T RH cells at 35 d.p.i. f , Cell numbers of lung CD8 + CD69 + NP 366-374 or CD8 + CD69 + PA 224-233 T RM , B GC or HA-specific B RM cells were enumerated from X31-infected Bcl6 fl/fl and Bcl6 ΔCD4ERT2 mice in the present of FTY720 (11-34 d.p.i.) at 35 d.p.i. In a-c and f , representative data were from at least two independent experiments (2-5 mice per group). In d-e , data were pooled from two independent experiments (3 mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Expressing, Injection, Two Tailed Test

    Both BCL6 and Bhlhe40 are required for optimal lung T RH responses. a-e , Bcl6 fl/fl or Bcl6 ΔT mice were infected with PR8. Representative dot plot ( a ), percentages ( b ) and cell numbers ( c ) of T RH or non-T RH in lung CD45 i.v. - total CD4 + or CD45 i.v. - influenza-specific CD4 + NP 311-325 T cells at 28 d.p.i. d-e , Representative dot plot ( d ) and percentage (top row) or cell numbers (bottom row) ( e ) of splenic total T FH or NP 311-325 T FH at 28 d.p.i. f , GSEA of the Bhlhe40 -associated genes in lung T RH (PD-1 Hi FR4 Hi ) and spleen T FH cells. g-k , Bhlhe40 fl/fl or Bhlhe40 ΔT mice were infected with PR8. g-i , Representative dot plot ( g ), percentages ( h ) or cell numbers ( i ) of influenza-specific NP 311-325 lung T RH , lung non-T RH , splenic T FH or splenic non-T FH cells at 28 d.p.i. j-k Representative dot plot ( j ) or percentages ( k ) of active caspase 3/7-FLICA + cells in lung NP 311-325 T RH or non-T RH cells at 28 d.p.i. In a-e and g-i , data were pooled from two independent experiments (3-4 mice per group). In j-k , representative data were from at least two independent experiments (4 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Both BCL6 and Bhlhe40 are required for optimal lung T RH responses. a-e , Bcl6 fl/fl or Bcl6 ΔT mice were infected with PR8. Representative dot plot ( a ), percentages ( b ) and cell numbers ( c ) of T RH or non-T RH in lung CD45 i.v. - total CD4 + or CD45 i.v. - influenza-specific CD4 + NP 311-325 T cells at 28 d.p.i. d-e , Representative dot plot ( d ) and percentage (top row) or cell numbers (bottom row) ( e ) of splenic total T FH or NP 311-325 T FH at 28 d.p.i. f , GSEA of the Bhlhe40 -associated genes in lung T RH (PD-1 Hi FR4 Hi ) and spleen T FH cells. g-k , Bhlhe40 fl/fl or Bhlhe40 ΔT mice were infected with PR8. g-i , Representative dot plot ( g ), percentages ( h ) or cell numbers ( i ) of influenza-specific NP 311-325 lung T RH , lung non-T RH , splenic T FH or splenic non-T FH cells at 28 d.p.i. j-k Representative dot plot ( j ) or percentages ( k ) of active caspase 3/7-FLICA + cells in lung NP 311-325 T RH or non-T RH cells at 28 d.p.i. In a-e and g-i , data were pooled from two independent experiments (3-4 mice per group). In j-k , representative data were from at least two independent experiments (4 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Two Tailed Test

    T cell-specific BCL6 or Bhlhe40 deficiency leads to impaired lung CD8 + memory and B cell immunity. a-d , Bcl6 fl/fl or Bcl6 ΔT mice were infected with PR8. a , Representative confocal images of lung iBALT at 28 d.p.i. Lung sections were stained with α-CD4 (red), α-B220 (green), α-GL7 (white) and DAPI (blue). Percentages of lung B GC ( b ), lung tissue HA-specific B RM or circulating HA-specific B MEM ( c ) and CD8 + CD69 + NP 366-374, or CD8 + CD69 + PA 224-233 T RM ( d ). e-g , Bhlhe40 fl/fl or Bhlhe40 ΔT mice were infected with PR8. e , Representative dot plot or cell numbers of lung tissue CD8 + T (top) or B cells (bottom) at 28 d.p.i. f , Cell numbers of NP-specific B RM or HA-specific B RM cells. g , Cell numbers of CD8 + CD69 + NP 366-374, or CD8 + CD69 + PA 224-233 T RM. . In a-d and f-g , representative data were from at least two independent experiments (4-5 mice per group). In e , data were pooled from two independent experiments (4 mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: T cell-specific BCL6 or Bhlhe40 deficiency leads to impaired lung CD8 + memory and B cell immunity. a-d , Bcl6 fl/fl or Bcl6 ΔT mice were infected with PR8. a , Representative confocal images of lung iBALT at 28 d.p.i. Lung sections were stained with α-CD4 (red), α-B220 (green), α-GL7 (white) and DAPI (blue). Percentages of lung B GC ( b ), lung tissue HA-specific B RM or circulating HA-specific B MEM ( c ) and CD8 + CD69 + NP 366-374, or CD8 + CD69 + PA 224-233 T RM ( d ). e-g , Bhlhe40 fl/fl or Bhlhe40 ΔT mice were infected with PR8. e , Representative dot plot or cell numbers of lung tissue CD8 + T (top) or B cells (bottom) at 28 d.p.i. f , Cell numbers of NP-specific B RM or HA-specific B RM cells. g , Cell numbers of CD8 + CD69 + NP 366-374, or CD8 + CD69 + PA 224-233 T RM. . In a-d and f-g , representative data were from at least two independent experiments (4-5 mice per group). In e , data were pooled from two independent experiments (4 mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Staining, Two Tailed Test

    T RH cells are required for the development of lung protective CD8 + T RM and B cell immunity. a-j , Bcl6 fl/fl or Bcl6 ΔCD4ERT2 mice were infected with PR8. a-e , Tamoxifen was administrated daily from 12-16 d.p.i. in the presence of daily FTY720 administration (11-34 d.p.i.). a , Schematics of experimental design. Cell numbers of B GC ( b ), HA-specific B RM ( c ), NP-specific B RM ( d ), e , CD8 + CD69 + NP 366-374 T RM or CD8 + CD69 + PA 224-233 T RM at 35 d.p.i. f-j , Tamoxifen was administrated daily from 21-25 d.p.i. in the presence of daily FTY720 administration (20-41 d.p.i.). f , Schematics of experimental design. Cell numbers of B GC ( g ), HA-specific B RM ( h ), NP-specific B RM ( i ), j , CD8 + CD69 + NP 366-374 T RM or CD8 + CD69 + PA 224-233 T RM at 42 d.p.i. k , Bcl6 fl/fl or Bcl6 ΔCD4ERT2 mice were infected with X31 strain (H3N2) of influenza. Tamoxifen was administrated daily from 12-16 d.p.i. in the presence of daily FTY720 administration (11-34 d.p.i.). Representative dot plot (top) and cell numbers (bottom) of X31 strain-specific B RM or cross-reactive HA-specific B RM (to H3N2 A/Uruguay/716/07 strain) at 35 d.p.i. l-m , Bcl6 fl/fl (n = 11) or Bcl6 ΔCD4ERT2 (n = 15) mice were infected with X31 and administered with tamoxifen from 12 to 16 d.p.i. Mice were re-challenged with PR8 at 42 d.p.i. in the presence of FTY720 (starting from 41d). l , Schematics of experimental design. m , Host mortality following PR8 challenge was monitored. In a-h and j-k , all data were pooled from two ( c, d, k, g, h and j ) or three ( b and e ) independent experiments (2-5 mice per group). In a-k, P values were calculated by unpaired two-tailed Student’s t-test. P value of survival study in m was calculated by Logrank test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: T RH cells are required for the development of lung protective CD8 + T RM and B cell immunity. a-j , Bcl6 fl/fl or Bcl6 ΔCD4ERT2 mice were infected with PR8. a-e , Tamoxifen was administrated daily from 12-16 d.p.i. in the presence of daily FTY720 administration (11-34 d.p.i.). a , Schematics of experimental design. Cell numbers of B GC ( b ), HA-specific B RM ( c ), NP-specific B RM ( d ), e , CD8 + CD69 + NP 366-374 T RM or CD8 + CD69 + PA 224-233 T RM at 35 d.p.i. f-j , Tamoxifen was administrated daily from 21-25 d.p.i. in the presence of daily FTY720 administration (20-41 d.p.i.). f , Schematics of experimental design. Cell numbers of B GC ( g ), HA-specific B RM ( h ), NP-specific B RM ( i ), j , CD8 + CD69 + NP 366-374 T RM or CD8 + CD69 + PA 224-233 T RM at 42 d.p.i. k , Bcl6 fl/fl or Bcl6 ΔCD4ERT2 mice were infected with X31 strain (H3N2) of influenza. Tamoxifen was administrated daily from 12-16 d.p.i. in the presence of daily FTY720 administration (11-34 d.p.i.). Representative dot plot (top) and cell numbers (bottom) of X31 strain-specific B RM or cross-reactive HA-specific B RM (to H3N2 A/Uruguay/716/07 strain) at 35 d.p.i. l-m , Bcl6 fl/fl (n = 11) or Bcl6 ΔCD4ERT2 (n = 15) mice were infected with X31 and administered with tamoxifen from 12 to 16 d.p.i. Mice were re-challenged with PR8 at 42 d.p.i. in the presence of FTY720 (starting from 41d). l , Schematics of experimental design. m , Host mortality following PR8 challenge was monitored. In a-h and j-k , all data were pooled from two ( c, d, k, g, h and j ) or three ( b and e ) independent experiments (2-5 mice per group). In a-k, P values were calculated by unpaired two-tailed Student’s t-test. P value of survival study in m was calculated by Logrank test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Two Tailed Test

    Lung CD4 + T cells deliver localized help for the development of tissue-resident B and CD8 + T cells. a-e , WT mice were infected with PR8 strain of influenza virus and treated with control (ctl) IgG or α-CD4 starting at 14 d.p.i. Mice were injected with α-CD45 intravenously (i.v.) 5 min before sacrifice at 42 d.p.i. a , Experimental scheme. b , Representative confocal images of iBALT in the lung. Lung sections were stained with α-CD4 (red), α-B220 (green) and DAPI (blue). c , Frequencies and cell number of influenza HA-specific B cells (HA-B) in the lung tissue (CD45 i.v. - B220 + GL7 - HA + ), lung blood vessels (CD45 i.v. + B220 + GL7 - HA + ) and spleen (B220 + GL7 - HA + ). d , Lung tissue CD8 + , CD8 + CD69 + or CD8 + CD69 + CD103 + NP 366-374 or PA 224-233 T RM cells were enumerated. e , Splenic CD8 + NP 366-374 or PA 224-233 memory cells (T MEM-SPL ) were enumerated. f-j , WT mice were infected with PR8 and treated with ctrl IgG or α-CD4 (starting at 14 d.p.i.) in the presence of daily injection of FTY-720 (starting at 13 d.p.i.). f , Schematic of experimental design. g , B GC cell numbers were enumerated by flow cytometry. h, i , Total HA-specific B cells (total HA-B) ( h ) or HA-specific tissue-resident memory B cells (HA-B RM : CD45 i.v. - B220 + GL7 - IgD - IgM - HA + CD38 + ) ( i ) were enumerated. j , Lung tissue CD8 + , CD8 + CD69 + or CD8 + CD69 + CD103 + NP 366-374 or PA 224-233 T RM cells were enumerated. k-n , WT mice were infected with PR8 and received ctl IgG, high or low dose of α-CD4. Cell number of B GC ( k ), HA-specific B RM ( l ) and NP-specific B RM cells ( m ) in the lung tissue. n , Lung tissue CD8 + , CD8 + CD69 + or CD8 + CD69 + CD103 + NP 366-374 T RM cells were enumerated. In b-e were the representative data from at least two independent experiments (4-5 mice per group). In g-h and j-n , data were pooled from two ( g, h and j ) or three ( k - n ) independent experiments (2-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test in c-j. P values in k-n were analyzed by one-way ANOVA.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Lung CD4 + T cells deliver localized help for the development of tissue-resident B and CD8 + T cells. a-e , WT mice were infected with PR8 strain of influenza virus and treated with control (ctl) IgG or α-CD4 starting at 14 d.p.i. Mice were injected with α-CD45 intravenously (i.v.) 5 min before sacrifice at 42 d.p.i. a , Experimental scheme. b , Representative confocal images of iBALT in the lung. Lung sections were stained with α-CD4 (red), α-B220 (green) and DAPI (blue). c , Frequencies and cell number of influenza HA-specific B cells (HA-B) in the lung tissue (CD45 i.v. - B220 + GL7 - HA + ), lung blood vessels (CD45 i.v. + B220 + GL7 - HA + ) and spleen (B220 + GL7 - HA + ). d , Lung tissue CD8 + , CD8 + CD69 + or CD8 + CD69 + CD103 + NP 366-374 or PA 224-233 T RM cells were enumerated. e , Splenic CD8 + NP 366-374 or PA 224-233 memory cells (T MEM-SPL ) were enumerated. f-j , WT mice were infected with PR8 and treated with ctrl IgG or α-CD4 (starting at 14 d.p.i.) in the presence of daily injection of FTY-720 (starting at 13 d.p.i.). f , Schematic of experimental design. g , B GC cell numbers were enumerated by flow cytometry. h, i , Total HA-specific B cells (total HA-B) ( h ) or HA-specific tissue-resident memory B cells (HA-B RM : CD45 i.v. - B220 + GL7 - IgD - IgM - HA + CD38 + ) ( i ) were enumerated. j , Lung tissue CD8 + , CD8 + CD69 + or CD8 + CD69 + CD103 + NP 366-374 or PA 224-233 T RM cells were enumerated. k-n , WT mice were infected with PR8 and received ctl IgG, high or low dose of α-CD4. Cell number of B GC ( k ), HA-specific B RM ( l ) and NP-specific B RM cells ( m ) in the lung tissue. n , Lung tissue CD8 + , CD8 + CD69 + or CD8 + CD69 + CD103 + NP 366-374 T RM cells were enumerated. In b-e were the representative data from at least two independent experiments (4-5 mice per group). In g-h and j-n , data were pooled from two ( g, h and j ) or three ( k - n ) independent experiments (2-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test in c-j. P values in k-n were analyzed by one-way ANOVA.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Injection, Staining, Flow Cytometry, Two Tailed Test

    Identification of a population of T FH -like cells in the lung tissue. WT ( a-e ) or IL-21-VFP reporter ( f ) mice were infected with PR8. a , tSNE plot of scRNAseq analysis of sorted lung CD45 i.v. - CD4 + CD44 Hi cells (pooled from 5 mice) at 28 d.p.i. b , Heat map of indicated genes in each cluster from scRNAseq data. c , Kinetics of the percentages of PD-1 Hi FR4 Hi population in lung tissue total CD4 + or influenza NP-specific (NP 311-325 ) CD4 + T cells. d , Expression of T FH cell-associated markers in lung total or influenza-specific PD-1 Hi FR4 Hi , PD-1 Low FR4 Low or splenic T FH (CD4 + CD44 Hi PD-1 + CXCR5 + ) cells at 28 d.p.i. e , Expression of IFN-γ, IL-17 or IL-4 by lung PD-1 Hi FR4 Hi , PD-1 Low FR4 Low or spleen T FH cells were identified by intracellular staining at 28 d.p.i . f , IL-21-VFP expression in lung CD4 + PD-1 Hi FR4 Hi , PD-1 Low FR4 Low or spleen T FH at 28 d.p.i. In c-f , representative plots, histograms and graphs were from at least two independent experiments (4 mice per group). P values in e and f were analyzed by one-way ANOVA.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Identification of a population of T FH -like cells in the lung tissue. WT ( a-e ) or IL-21-VFP reporter ( f ) mice were infected with PR8. a , tSNE plot of scRNAseq analysis of sorted lung CD45 i.v. - CD4 + CD44 Hi cells (pooled from 5 mice) at 28 d.p.i. b , Heat map of indicated genes in each cluster from scRNAseq data. c , Kinetics of the percentages of PD-1 Hi FR4 Hi population in lung tissue total CD4 + or influenza NP-specific (NP 311-325 ) CD4 + T cells. d , Expression of T FH cell-associated markers in lung total or influenza-specific PD-1 Hi FR4 Hi , PD-1 Low FR4 Low or splenic T FH (CD4 + CD44 Hi PD-1 + CXCR5 + ) cells at 28 d.p.i. e , Expression of IFN-γ, IL-17 or IL-4 by lung PD-1 Hi FR4 Hi , PD-1 Low FR4 Low or spleen T FH cells were identified by intracellular staining at 28 d.p.i . f , IL-21-VFP expression in lung CD4 + PD-1 Hi FR4 Hi , PD-1 Low FR4 Low or spleen T FH at 28 d.p.i. In c-f , representative plots, histograms and graphs were from at least two independent experiments (4 mice per group). P values in e and f were analyzed by one-way ANOVA.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Expressing, Staining

    Impaired T RH responses following B cell depletion, iBALT disruption or Bhlhe40 deficiency. a-c , WT ( a and c ) or IL-21-VFP reporter ( b ) mice were infected with PR8 and received with ctl IgG, α-CD20 or LTβR-Ig weekly in the present of FTY-720 (Experimental scheme in Fig 5 . a.). a , The efficiency of B cell depletion in the lung. b , Representative confocal images of IL-21-expressing cells in iBALT. c , Cell numbers of lung tissue B cells in mice received with ctl-IgG or LTβR-Ig. d-g , Bhlhe40 fl/fl or Bhlhe40 ΔT mice were infected with PR8. Representative dot plot ( d ), percentages (top) or cell numbers (bottom) ( e ) of total lung T RH , non-T RH , splenic T FH or splenic non-T FH at 28 d.p.i. f , Representative dot plot or cell numbers of lung tissue or splenic CD4 + T cells at 28 d.p.i. g , Cell numbers of lung or splenic influenza-NP 311-325 CD4 + T cell at 28 d.p.i. Data were pooled from two independent experiments (3-4 mice per group). P value were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Impaired T RH responses following B cell depletion, iBALT disruption or Bhlhe40 deficiency. a-c , WT ( a and c ) or IL-21-VFP reporter ( b ) mice were infected with PR8 and received with ctl IgG, α-CD20 or LTβR-Ig weekly in the present of FTY-720 (Experimental scheme in Fig 5 . a.). a , The efficiency of B cell depletion in the lung. b , Representative confocal images of IL-21-expressing cells in iBALT. c , Cell numbers of lung tissue B cells in mice received with ctl-IgG or LTβR-Ig. d-g , Bhlhe40 fl/fl or Bhlhe40 ΔT mice were infected with PR8. Representative dot plot ( d ), percentages (top) or cell numbers (bottom) ( e ) of total lung T RH , non-T RH , splenic T FH or splenic non-T FH at 28 d.p.i. f , Representative dot plot or cell numbers of lung tissue or splenic CD4 + T cells at 28 d.p.i. g , Cell numbers of lung or splenic influenza-NP 311-325 CD4 + T cell at 28 d.p.i. Data were pooled from two independent experiments (3-4 mice per group). P value were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Expressing, Two Tailed Test

    Lung “local” CD4 T cell help for the development robust memory CD8 + and B cell immunity. WT mice were infected with influenza PR8 ( a ) or X31 ( b-e ) and treated with control IgG or α-CD4 (starting at 14 d.p.i.) in the presence of daily FTY720 (starting at 13 d.p.i.). a , Blood lymphocytes in the PBS or FTY720 administrated mice. b-e , Numbers of lung B GC cells ( b ), total HA specific B cells ( c ), HA-specific B RM ( d ), and total CD8 + NP 366-374 memory T cells, CD8 + CD69 + NP 366-374 T RM or CD8 + CD69 + CD103 + NP 366-374 T RM cells ( e). f-h , WT mice were infected with PR8 and received with ctl IgG, low or high dose of α-CD4 (starting at 14 d.p.i.). Experimental scheme ( f ), representative dot plot of blood lymphocyte population ( g ) and lung lymphocyte population ( h ). Representative data were from at least two independent experiments (4-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Lung “local” CD4 T cell help for the development robust memory CD8 + and B cell immunity. WT mice were infected with influenza PR8 ( a ) or X31 ( b-e ) and treated with control IgG or α-CD4 (starting at 14 d.p.i.) in the presence of daily FTY720 (starting at 13 d.p.i.). a , Blood lymphocytes in the PBS or FTY720 administrated mice. b-e , Numbers of lung B GC cells ( b ), total HA specific B cells ( c ), HA-specific B RM ( d ), and total CD8 + NP 366-374 memory T cells, CD8 + CD69 + NP 366-374 T RM or CD8 + CD69 + CD103 + NP 366-374 T RM cells ( e). f-h , WT mice were infected with PR8 and received with ctl IgG, low or high dose of α-CD4 (starting at 14 d.p.i.). Experimental scheme ( f ), representative dot plot of blood lymphocyte population ( g ) and lung lymphocyte population ( h ). Representative data were from at least two independent experiments (4-5 mice per group). P values were calculated by unpaired two-tailed Student’s t-test.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Two Tailed Test

    Lung PD-1 Hi FR4 Hi cells are tissue resident a , WT mice were infected with PR8. The expression of CD69, CXCR6 and Bhlhe40 in lung CD4 + PD-1 Hi FR4 Hi or CD4 + PD-1 Low FR4 Low NP 311-325 T cells or splenic T FH cells at 28 d.p.i. b-e , CD45.1 + (Host) or CD45.1 + CD45.2 + (Partner) WT mice were infected with PR8. Parabiosis surgery was performed at 21 d.p.i. Mice were sacrificed 14 days later for analysis. b , Schematics of parabiosis experiments. c , Composition of Host-derived or Partner-derived CD4 + T cells in the spleens of the parabionts. d , Frequencies of Host-derived or Partner-derived cells in the lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low total CD4 + T cell compartment. e , Frequencies of Host-derived or Partner-derived cells in influenza-specific lung CD4 + PD-1 Hi FR4 Hi or CD4 + PD-1 Low FR4 Low NP 311-325 T cell compartment. In a , the representative histograms were from at least two independent experiments (3-4 mice per group). Parabiosis data were pooled from two different experiments (two pairs per experiment). P values in d and e were analyzed by one-way ANOVA.

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Lung PD-1 Hi FR4 Hi cells are tissue resident a , WT mice were infected with PR8. The expression of CD69, CXCR6 and Bhlhe40 in lung CD4 + PD-1 Hi FR4 Hi or CD4 + PD-1 Low FR4 Low NP 311-325 T cells or splenic T FH cells at 28 d.p.i. b-e , CD45.1 + (Host) or CD45.1 + CD45.2 + (Partner) WT mice were infected with PR8. Parabiosis surgery was performed at 21 d.p.i. Mice were sacrificed 14 days later for analysis. b , Schematics of parabiosis experiments. c , Composition of Host-derived or Partner-derived CD4 + T cells in the spleens of the parabionts. d , Frequencies of Host-derived or Partner-derived cells in the lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low total CD4 + T cell compartment. e , Frequencies of Host-derived or Partner-derived cells in influenza-specific lung CD4 + PD-1 Hi FR4 Hi or CD4 + PD-1 Low FR4 Low NP 311-325 T cell compartment. In a , the representative histograms were from at least two independent experiments (3-4 mice per group). Parabiosis data were pooled from two different experiments (two pairs per experiment). P values in d and e were analyzed by one-way ANOVA.

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, Expressing, Derivative Assay

    Transcriptional profiling reveals PD-1 Hi FR4 Hi cells exhibit both T FH and T RM gene signatures. a-f , WT mice were infected with PR8. Lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low CD4 + T cells and splenic T FH or non-T FH cells were sorted following exclusion of GITR Hi Treg cells and RNA-seq analysis was performed at 28 d.p.i. a , Heatmap expression of differentially expressed genes among lung PD-1 Hi FR4 Hi , PD-1 Low FR4 Low CD4 + T cells and splenic T FH or non-T FH cells. b , Principle component analysis of RNA-seq data of lung PD-1 Hi FR4 Hi , PD-1 Low FR4 Low CD4 + T cells and splenic T FH or non-T FH cells. c , Volcano plot of RNA-seq analysis of lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low CD4 + T cells. d , GSEA of the core T FH signature genes in lung CD4 + PD-1 Hi FR4 Hi and CD4 + PD-1 Low FR4 Low cells. e , Volcano plot of RNA-seq analysis on lung PD-1 Hi FR4 Hi CD4 + T and splenic T FH cells. f , GSEA of the core tissue-residency signature genes of T RM cells in lung PD-1 Hi FR4 Hi and splenic T FH cells. g-h , WT mice were infected with PR8. Lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low CD4 + T cells and splenic T FH cells were sorted at 28 d.p.i. Nanostring analysis on 560 immune-associated genes was performed. The expression of T FH -associated genes ( g ) or tissue-residency associated genes ( h ) in the three cell populations was depicted. For RNA-seq, data were from duplicates of pooled samples (n = 15). For nanostring analysis, data were from pooled samples (n = 10).

    Journal: bioRxiv

    Article Title: Tissue-resident CD4+ T helper cells assist protective respiratory mucosal B and CD8+ T cell memory responses

    doi: 10.1101/2020.02.28.970400

    Figure Lengend Snippet: Transcriptional profiling reveals PD-1 Hi FR4 Hi cells exhibit both T FH and T RM gene signatures. a-f , WT mice were infected with PR8. Lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low CD4 + T cells and splenic T FH or non-T FH cells were sorted following exclusion of GITR Hi Treg cells and RNA-seq analysis was performed at 28 d.p.i. a , Heatmap expression of differentially expressed genes among lung PD-1 Hi FR4 Hi , PD-1 Low FR4 Low CD4 + T cells and splenic T FH or non-T FH cells. b , Principle component analysis of RNA-seq data of lung PD-1 Hi FR4 Hi , PD-1 Low FR4 Low CD4 + T cells and splenic T FH or non-T FH cells. c , Volcano plot of RNA-seq analysis of lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low CD4 + T cells. d , GSEA of the core T FH signature genes in lung CD4 + PD-1 Hi FR4 Hi and CD4 + PD-1 Low FR4 Low cells. e , Volcano plot of RNA-seq analysis on lung PD-1 Hi FR4 Hi CD4 + T and splenic T FH cells. f , GSEA of the core tissue-residency signature genes of T RM cells in lung PD-1 Hi FR4 Hi and splenic T FH cells. g-h , WT mice were infected with PR8. Lung PD-1 Hi FR4 Hi or PD-1 Low FR4 Low CD4 + T cells and splenic T FH cells were sorted at 28 d.p.i. Nanostring analysis on 560 immune-associated genes was performed. The expression of T FH -associated genes ( g ) or tissue-residency associated genes ( h ) in the three cell populations was depicted. For RNA-seq, data were from duplicates of pooled samples (n = 15). For nanostring analysis, data were from pooled samples (n = 10).

    Article Snippet: PR8-NP protein was purchased from Sino Biological.

    Techniques: Mouse Assay, Infection, RNA Sequencing Assay, Expressing

    Heterosubtypic protection against H1 virus by anti–MHCII-MIX vaccination is independent of T cells. ( A and B ) BALB/c mice ( n = 7 per group) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines. Sera from mice of each group were harvested 2 wk after the third vaccination and transferred i.v. to naive mice ( n = 10 per group) before a lethal challenge with 5 × LD 50 PR8. Mice were monitored for weight loss (A) and survival (B). The results are representative of two independent experiments. ( C and D ) BALB/c mice ( n = 10) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines and subsequently were injected every other day from day 12 after the third vaccination with depleting mAbs against CD4 and CD8 T cells or isotype-matched control mAbs. A 5 × LD 50 lethal dose of PR8 was administered at day 14, and the mice were monitored for weight loss (C) and survival (D). Efficient depletion was confirmed by staining of splenocytes. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza

    doi: 10.4049/jimmunol.1701088

    Figure Lengend Snippet: Heterosubtypic protection against H1 virus by anti–MHCII-MIX vaccination is independent of T cells. ( A and B ) BALB/c mice ( n = 7 per group) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines. Sera from mice of each group were harvested 2 wk after the third vaccination and transferred i.v. to naive mice ( n = 10 per group) before a lethal challenge with 5 × LD 50 PR8. Mice were monitored for weight loss (A) and survival (B). The results are representative of two independent experiments. ( C and D ) BALB/c mice ( n = 10) were immunized three times (weeks 0, 4, and 7) with 25 μg of DNA/EP of the indicated vaccines and subsequently were injected every other day from day 12 after the third vaccination with depleting mAbs against CD4 and CD8 T cells or isotype-matched control mAbs. A 5 × LD 50 lethal dose of PR8 was administered at day 14, and the mice were monitored for weight loss (C) and survival (D). Efficient depletion was confirmed by staining of splenocytes. * p

    Article Snippet: ELISA 96-well plates (Costar 3590) were coated with one of the following recombinant influenza HA proteins: PR8 (11684-V08H; Sino Biological), A/Hong Kong/483/97 (H5N1) (11689-V08H; Sino Biological), A/northern shoveler/California/HKWF115/07 (H6N1) (MBS434125; MyBioSource, San Diego, CA, or 11723-V08H; Sino Biological), A/pintail duck/Alberta/114/1979 (H8N4) (11722-V08H; Sino Biological), A/Hong Kong/1073/99 (H9N2) (11229-V08H; Sino Biological), A/duck/Yangzhou/906/2002 (H11N2) (11705-V08H; Sino Biological), A/black-headed gull/Netherlands/1/00 (H13N8) (11721-V08H; Sino Biological), A/Shanghai/1/2013 (H7N9) (40104-V08B; Sino Biological), or A/Hong Kong/1/1968 (H3N2) (40116-V08B; Sino Biological).

    Techniques: Mouse Assay, Injection, Staining

    Vaccination with anti–MHCII-MIX confers heterosubtypic protection against H1 influenza viruses not included in the vaccine mixture. ( A – F ) BALB/c mice ( n = 6 per group) were immunized three times (weeks 0, 4, and 12, arrows) with 25 μg of DNA/EP of the indicated vaccines. As positive controls, anti–MHCII-H1(PR8) and anti–MHCII-H1(Cal07) were included. Serum IgG Abs were measured against PR8 (A) or Cal07 (B) in ELISAs (mean ± SEM). Microneutralization assays were performed on sera harvested 2 wk after the third vaccination with PR8 (C) and Cal07 (D). The dashed line indicates 50% threshold for positive neutralization. Two weeks after the third vaccination (above), mice were challenged with a lethal dose of Cal07 and monitored for weight loss (± SEM) (E) and survival (F). ( G and H ) In a new experiment, mice were vaccinated at weeks 0, 4, and 12 (as above) and challenged with a lethal dose of influenza PR8 2 wk after the third vaccination ( n = 10 per group [black], n = 6 per group [gray]). Mice were monitored for weight loss (± SEM) (G) and survival (H). * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza

    doi: 10.4049/jimmunol.1701088

    Figure Lengend Snippet: Vaccination with anti–MHCII-MIX confers heterosubtypic protection against H1 influenza viruses not included in the vaccine mixture. ( A – F ) BALB/c mice ( n = 6 per group) were immunized three times (weeks 0, 4, and 12, arrows) with 25 μg of DNA/EP of the indicated vaccines. As positive controls, anti–MHCII-H1(PR8) and anti–MHCII-H1(Cal07) were included. Serum IgG Abs were measured against PR8 (A) or Cal07 (B) in ELISAs (mean ± SEM). Microneutralization assays were performed on sera harvested 2 wk after the third vaccination with PR8 (C) and Cal07 (D). The dashed line indicates 50% threshold for positive neutralization. Two weeks after the third vaccination (above), mice were challenged with a lethal dose of Cal07 and monitored for weight loss (± SEM) (E) and survival (F). ( G and H ) In a new experiment, mice were vaccinated at weeks 0, 4, and 12 (as above) and challenged with a lethal dose of influenza PR8 2 wk after the third vaccination ( n = 10 per group [black], n = 6 per group [gray]). Mice were monitored for weight loss (± SEM) (G) and survival (H). * p

    Article Snippet: ELISA 96-well plates (Costar 3590) were coated with one of the following recombinant influenza HA proteins: PR8 (11684-V08H; Sino Biological), A/Hong Kong/483/97 (H5N1) (11689-V08H; Sino Biological), A/northern shoveler/California/HKWF115/07 (H6N1) (MBS434125; MyBioSource, San Diego, CA, or 11723-V08H; Sino Biological), A/pintail duck/Alberta/114/1979 (H8N4) (11722-V08H; Sino Biological), A/Hong Kong/1073/99 (H9N2) (11229-V08H; Sino Biological), A/duck/Yangzhou/906/2002 (H11N2) (11705-V08H; Sino Biological), A/black-headed gull/Netherlands/1/00 (H13N8) (11721-V08H; Sino Biological), A/Shanghai/1/2013 (H7N9) (40104-V08B; Sino Biological), or A/Hong Kong/1/1968 (H3N2) (40116-V08B; Sino Biological).

    Techniques: Mouse Assay, Neutralization

    Characterization of vaccine proteins. ( A ) The MHCII-targeted vaccine proteins consist of a targeting unit (scFv) that is specific for MHC class II (I), a dimerization unit containing a shortened hinge and C H 3 from human γ3 (II), and an antigenic unit encoding one of the six indicated HAs (III). Nontargeted controls have been prepared by replacement of the targeting unit with an scFv that is specific for the hapten NIP. ( B ) Supernatants from HEK 293E cells transfected with the DNA plasmids encoding the indicated vaccines were analyzed in sandwich ELISAs. An MHCII-targeted vaccine encoding HA from PR8 (anti–MHCII-H1) was included as positive control.

    Journal: The Journal of Immunology Author Choice

    Article Title: Simultaneous Targeting of Multiple Hemagglutinins to APCs for Induction of Broad Immunity against Influenza

    doi: 10.4049/jimmunol.1701088

    Figure Lengend Snippet: Characterization of vaccine proteins. ( A ) The MHCII-targeted vaccine proteins consist of a targeting unit (scFv) that is specific for MHC class II (I), a dimerization unit containing a shortened hinge and C H 3 from human γ3 (II), and an antigenic unit encoding one of the six indicated HAs (III). Nontargeted controls have been prepared by replacement of the targeting unit with an scFv that is specific for the hapten NIP. ( B ) Supernatants from HEK 293E cells transfected with the DNA plasmids encoding the indicated vaccines were analyzed in sandwich ELISAs. An MHCII-targeted vaccine encoding HA from PR8 (anti–MHCII-H1) was included as positive control.

    Article Snippet: ELISA 96-well plates (Costar 3590) were coated with one of the following recombinant influenza HA proteins: PR8 (11684-V08H; Sino Biological), A/Hong Kong/483/97 (H5N1) (11689-V08H; Sino Biological), A/northern shoveler/California/HKWF115/07 (H6N1) (MBS434125; MyBioSource, San Diego, CA, or 11723-V08H; Sino Biological), A/pintail duck/Alberta/114/1979 (H8N4) (11722-V08H; Sino Biological), A/Hong Kong/1073/99 (H9N2) (11229-V08H; Sino Biological), A/duck/Yangzhou/906/2002 (H11N2) (11705-V08H; Sino Biological), A/black-headed gull/Netherlands/1/00 (H13N8) (11721-V08H; Sino Biological), A/Shanghai/1/2013 (H7N9) (40104-V08B; Sino Biological), or A/Hong Kong/1/1968 (H3N2) (40116-V08B; Sino Biological).

    Techniques: Transfection, Positive Control